Abstract

Recent studies on the modularity of mitogen-activated protein kinases show how redesigning
'surface patches' on a protein can change the topology of a signaling network.

Minireview

In cells, protein-protein interaction domains control the organization of multiprotein
complexes in signal transduction networks, thereby determining the responses of cells
to many different stimuli [1]. Such domains are generally defined as independently folded structural modules that
can bind a protein ligand or a peptide motif. There are at least 81 defined protein-interaction
domains in eukaryotic cells that control the organization and responses of signaling
networks [2]. Even a given domain can have significant complexity and be used repeatedly in different
contexts. For example, more than 120 Srchomology 2 (SH2) domains - which recognize
phosphotyrosines - are encoded in the human genome. Each SH2 domain has amino acid
variations that alter the sequence context within which it recognizes a phosphotyrosine
residue. In higher eukaryotes especially, a single protein is typically composed of
multiple domains, and so the ability to reconfigure the repertoire of domain composition
and position within a protein provides a powerful mechanism for reconfiguring the
architecture of signaling networks both in evolution and by design engineering [3-5].

Although domain-wiring models, defined by domain-dependent protein metrications, have
proved to be particularly valuable in predicting protein interactions within complex
networks, they best describe how the primary backbone of the network is laid out.
The high-fidelity choice of interaction partner can only be partly explained by domain-wiring.
For instance, a degree of interaction specificity can be controlled by variation within
the domain itself, as evidenced by the 120 or so different members of the SH2-domain
family. However, it is clear that in many cases the specificity of a protein interaction
cannot lie entirely with the interacting amino acids in the binding site, and a degree
of 'fine-tuning' of specificity occurs elsewhere in the protein. The recent work of
Mody et al. [6] published in Nature Cell Biology helps shed light on how the modularity of two yeast mitogen-activated protein kinases
(MAPKs) establishes a capability for altering the specificity of interaction and,
therefore, for changing the topology of a signaling network.

The modular nature of MAPKs

MAPKs are relatively small proteins with an average mass of around 40 kDa. The three-dimensional
structures of several MAPKs are known and show them to be compact globular proteins
[7,8]. MAPKs are serine-threonine kinases that phosphorylate diverse transcription factors,
intracellular enzymes and cytoskeletal proteins to control gene expression and the
physiological program of the cell. They are activated by MAPK kinases (MKKs) via the
phosphorylation of a threonine and a tyrosine in a conserved Thr-X-Tyr motif on the
'phosphorylation lip' of the kinase domain, and are inactivated by specific phosphatases
that remove these phosphate groups. In addition, MAPKs often bind specific scaffold
proteins such as Ste5 in yeast and KSR in mammalian cells [9,10]. In response to a particular signal (such as pheromone signaling), scaffold proteins
such as Ste5 bind and organize specific components of a 'MAPK cascade' - MAPK kinase
kinases (MKKKs), MKKs and MAPKs - in such a way that they interact effectively with
each other [9,10]. Each different MAPK must therefore interact with high specificity with multiple
proteins so that MAPK signaling networks responding to different stimuli can be formed
and regulated.

The specific interactions MAPKs make with their cognate MKKs, substrates, scaffolds
and phosphatases contribute significantly to pathway specificity, and involve a docking
groove found in all MAPKs that contains a basic region and a hydrophobic region and
binds the hydrophobic docking-peptide motif φA-X-φB (where φA and φB are hydrophobic residues - Leu, Ile or Val) [11-13]. However, given the relative conservation of docking-groove amino acid sequence among
MAPKs, it is unlikely that the docking groove and the cognate binding motifs are the
only mechanism for controlling the specific interaction of MAPKs with their many ligands.

The recent work of Mody et al. [6] provides a significant advance beyond the docking groove in our understanding of
MAPK modularity and the determinants of its interaction with other proteins. These
investigators examined the sequence alignments of multiple yeast, human and plant
orthologs of Saccharomyces cerevisiae MAPKs. Focusing on Fus3 and Hog1, S. cerevisiae orthologs of the mammalian MAPKs ERK1/2 and p38, respectively, they hypothesized that
variable residues in particular surface regions or 'patches' in the two proteins could
contribute to the different activation and substrate specificities of Fus3 and Hog1.
Fus3 is activated by the MKK Ste7 and phosphorylates substrates such as the cell-cycle
arrest mediator Far1 in response to mating pheromone. In contrast, Hog1 is activated
by the MKK Pbs2 in response to hyperosmolar shock and phosphorylates several transcription
factors, including Hot1 and Sko1, thus initiating a response to osmolyte imbalance.
Mody et al. investigated the significance of the sequence patch in controlling the specificities
of Fus3 and Hog1 for their upstream MKKs and downstream transcription-factor substrates
by constructing kinases containing different combinations of amino acids from the
Fus3 and Hog1 patches (Figure 1). The chimeric proteins were expressed in appropriate genetic backgrounds in S. cerevisiae and tested for their ability to signal pheromone- or hyperosmolar-stimulated responses.

Figure 1. The responses to pheromone and sorbitol in the presence of different Fus3 and Hog1
hybrid proteins. (a) The hybrids of Fus3 and Hog1 are shown on the left. Capital letters ABCDEF (black)
each represent a segment of Fus3, while the lower-case letters abcdef (red) each represent
a segment of Hog1. The relative responses to pheromone and sorbitol were measured
using a FUS1 promoter-driven reporter gene to detect Fus3 activity (horizontal blue bars). Plasmids
bearing the hybrid genes were introduced into cells deleted for endogenous Fus3 (fus3Δ) and the MAPK Kss1 (kss1Δ), an alternative target for Ste5 activation. Mating activity is scored from +++ to
- (none). The lower panel in (a) shows the crossover response in which sorbitol activates
the FUS1-driven reporter gene when there is high-copy expression of the ABcdEF hybrid. The
Δ symbol indicates that the response was maintained in a Ste7-deleted background.
(b) The relative responses to pheromone and sorbitol were measured using a STL1 promoter-driven reporter gene to detect Hog1 activity (represented by blue bars).
Relative efficiency of growth on sorbitol is scored from +++ to -. Data in (a) and
(b) are from [6]. (c) Model modified from [6] depicting the ability of different sequence patches in Fus3/Hog1 hybrids to regulate
the pheromone and osmolyte activation of hybrid MAPKs.

Mody et al. [6] used six different segments of Fus3 and Hog1 in the combinations shown in Figure
1. The segments 'B' (or 'b') and 'F' (or 'f') contain the docking-groove sequences.
The BF segments from Fus3 were effective in maintaining the mating-pheromone response
mediated by the upstream MKK Ste7, and the bf segments from Hog1 were similarly effective
in maintaining the sorbitol hyperosmolar response (Figure 1a and 1b, respectively). These effects were most clear-cut when the chimeric MAPKs were expressed
from low-copy-number plasmids, which is more representative of their physiological
levels. When high-copy plasmids for high protein expression were used, there is significant
crossover, and responses to both pheromone and sorbitol were seen with each MAPK.
This is due to the high protein expression enabling lower-affinity interactions to
occur to a much greater extent.

Notably, cells expressing ABcdEF or ABcdeF had constitutive Fus3 activity. This is
particularly interesting because replacing Thr and Tyr in the Thr-X-Tyr activation-loop
motif with phosphomimetics does not activate Fus3 or Hog1. These results suggest that
the CD segments have a role in controlling the inactive state of Fus3 and that substitution
with the cd region of Hog1 relieves this inhibition. The three hybrids ABcdEF, ABcdeF
and aBcdeF showed Fus3 activity in response to sorbitol, even when Ste7 (the Fus3
MKK) and Hog1 were deleted. That result indicated a direct activation of the chimeric
protein by Pbs2, the MKK for Hog1, which was now able to recognize Fus3. This is particularly
telling, because these three hybrids encode the docking-groove BF segments of Fus3,
and it implies that segments A/a, C/c, D/d and E/e in Fus3 and Hog1 make significant
contributions to recognition by their cognate MKKs.

Figure 1c summarizes some of the salient findings from the hybrid analysis. These indicate
that an aBCDEF hybrid produces only a low-level mating response, thus implicating
segment A in the interaction of Fus3 with Far1. This is not too surprising, as segment
A/a includes the ATP-binding pocket and includes residues involved in substrate recognition.
Segment 'd' is important for transducing a hyperosmolar response to either pheromone
or sorbitol. It is required, although not sufficient, for activation of hybrid MAPKs
by sorbitol at low-copy expression. Segment d has a significant deletion relative
to D, with a somewhat neutral drift in the amino acid differences in D, suggesting
that the insert might be significant for the selectivity of Fus3 for Ste7 and Far1
in the mating response. Overall, the hybrid analysis shows that the different sequence
patches in A/a, C/c, D/d and E/e play significant roles in specificity in addition
to the roles played by the docking groove and activation loop (comprising segments
B/b and F/f).

Engineering MAPK signaling dynamics

Switching or modification of specific sequences on the MAPK surface enables the generation
of promiscuous enzymes that respond to multiple activators and act on multiple substrates,
the evolution of new specificities within signaling networks, and the engineering
of MAPK interactions to rewire network behavior. The identification by Mody et al. [6] of regions outside the docking groove that support interaction specificity expands
the ability to engineer MAPKs to have new functions. Thus, engineering these sequence
patches as well as the docking groove will enable the development of MAPKs with unique
connections for upstream activators, downstream substrates, inactivating phosphatases
and the scaffolds that organize the MAPK signaling complexes. Such specificity modifications
could be engineered in combination with scaffold modifications to allow assembly of
MAPK cascades that modulate positive- and negative-feedback loops controlling duration
and magnitude of activation, sensitivity of the system to specific stimuli, and the
ability to tune the system [14,15].

The modular nature of MAPKs and their scaffolds allows rational design principles
to be used to build synthetic responses for therapeutic uses. For example, one can
imagine a surface receptor expressed in vascular sentinel cells that binds a specific
disease-related biomarker released into the bloodstream that, in turn, activates a
synthetic MAPK system and sounds the alarm for early diagnosis and therapeutic intervention.
The extensive and growing knowledge base for designing synthetic MAPKs and scaffolds
suggests that such ideas are probably already in the making.